Morphology and growth potential of stromal cell cultures derived from human endometrium

Summary Propagable cell cultures derived from human endometrial tissue were determined to contain cells predominantly of stromal cell origin based on their morphologic resemblance to endometrial stromal cells. These features included nexi, solitary cilia, and predecidual cytology. In addition to morphology the cell cultures retained a normal karyotype and responded to steroid hormones as evidenced by cellular aggregation. The stromal cells were evaluated for a variety of characteristics associated with transformed cells and seemed to be biologically normal without neoplastic phenotypes. Growth potential of the stromal cell cultures was also characterized in normal maintenance medium, in nutritionally depleted medium with reduced levels of calcium or serum, and in medium with increased levels of serum. The prolonged survival of the stromal cells in vitro coupled with the retention of in vivo characteristics and an absence of neoplastic phenotype provides a human cell system that is amenable to a variety of long-term experimental analyses. This work was supported by contract CP75956 and grant CA31733 from the National Cancer Institute. B. Hugh Dorman was the recipient of a predoctoral scholarship from the Chemical Industry Institute of Toxicology. Jill M. Siegfried was supported by National Research Service Award CA09156. David G. Kaufman is the recipient of a Research Career Development Award (CA00431) from the National Cancer Institute.     

Transformed phenotype conferred to NIH/3T3 cells by ectopic expression of heparin-binding growth factor 1/acidic fibroblast growth factor

Summary Heparin-binding growth factor 1 (HBGF-1), also known as acidic fibroblast growth factor, is a potent mitogen and angiogenic factor found in tissues such as brain, kidney and heart. The genomic and cDNA sequences indicate that HBGF-1 does not have a typical signal peptide sequence. HBGF-1 was shown to be localized to the extracellular matrix of cardiac myocytes, but the mechanism of secretion is not presently known. We have cloned the HBGF-1 cDNA which allowed us to directly test the biological activity, mechanism of secretion and transforming potential of the recombinant protein. A previous report showed that the truncated HBGF-1 confers partial transformed phenotype to the recipient fibroblasts. However, expression of full-length HBGF-1 has not been reported. The HBGF-1 coding sequence was cloned into the retroviral expression vector, SVX, and transfected into NIH/3T3 cells. Transfectants expressing full-length HBGF-1 protein at high levels form foci and grow to a higher cell density than the parental NIH/3T3 cells. Western blotting analysis showed that the recombinant HBGF-1 is a unique band of approximately 20 kDa and can be detected in the cell homogenate but not in the conditioned medium. NIH/3T3 cells were conferred anchorage independence when HBGF-1 was provided exogenously. We showed the transformed cells are capable of growing on soft agar even in the absence of exogenously-provided HBGF-1. Transfected cells expressing HBGF-1 also induced tumor formation when injected into nude mice. Thus, NIH/3T3 cells acquired a full spectrum of transformed phenotype when full length HBGF-1 was expressed at high levels. This work was supported by grants from the National Cancer Institute (RO1 CA45611), The March of Dimes Birth Defects Foundation (No. 6-549) and The Ohio Cancer Research Associates, Inc. I.-M.C. is a recipient of The Research Career Development Award (KO4 CA01369) from the National Institutes of Health.     

Histochemical identification of cultured cells from human endometrium

Summary Histochemical techniques have been applied to the identification of cell types cultured from human endometrium. Previous work from this laboratory characterized two principtal cell types found in cultures of endometrium: a mature epithelial cell and another cell which was classified as the endometrial stromal cell based on light and electron microscopy. In this report we compare the histochemical staining of endometrial tissue in frozen sections to that of cultured cells. These results confirm the epithelial and stromal nature of the respective cell types. Several markers were found that could distinguish between cells of epithelial and stromal origin. The enzymes alkaline phosphatase, γ-glutamyltranspeptidase, peroxidase, and β-glucuronidase were localized in glandular and surface epithelia in frozen sections and in colonies of epithelial cells in culture. Stroma in frozen sections and cultured stromal cells contained leucine aminopeptidase and fibronectin. Epithelial sections and in culture could also be distinguished from cells of stromal origin by preferential binding of lotus and peanut lectin. Several other markers were found in both endometrial epithelium and stroma. J. M. S. was recipient of National Research Service Award CA09156 (National Cancer Institute); K. G. N. was recipient of National Research Service Award ES07017 (National Institute of Environmental Health Sciences); and D. G. K. was recipient of Research Career Development Award CA00431 from the National Cancer Institute, Bethesda, MD. Supported by Grant CA 31733 from the National Cancer Institute, Bethesda, MD.     

Growth of human-human hybridomas in serum-free media enhances antibody secretion

Summary Human-human hybridomas derived from fusing lymph node lymphocytes with UC 729-6 were adapted to grow in commercially available serum-free medium and were compared with serum-supplemented [10% fetal bovine serum (FBS)] cultures. Over a 6-d period, no significant changes occurred in the growth of the cells in 10% FBS or serum-free medium. In cultures supplemented with 10% FBS more total proteins were secreted than in serum-free cultures. However, there was an enhanced secretion of three- to four-fold of both immunoreactive human IgG and IgM under serum-free conditions compared to serum-supplemented conditions. Serum-free conditions may provide the appropriate milieu for the increased level of Ig secretion from human hybridomas derived from UC 729-6 in that there are no inhibitors that may be present in serum. The work described in this report was partially supported by grants from the National Institutes of Health (CA 32047, CA 37497), Bethesda, MD, and the University of California Cancer Research Coordinating Committee. R.E.P. was a postdoctoral fellow supported by the UCSD Cancer Center and M.C.G. was the recipient of an NIH New Investigator Research Award. A.M. was a visiting scientist from the Medical Academy of Bulgaria, Sofia.     

ESTDAB: a collection of immunologically characterised melanoma cell lines and searchable databank

Cancer immunologists working in humans often require panels of tumour cell lines expressing different combinations of HLA alleles and other characteristics. Sources of cell lines are usually large cell banks carrying little immunologically relevant information on the available cells and limited numbers of different lines from the same type of tumour. Access to cells with desired combinations of characteristics is, therefore, difficult. Here, we describe an interactive database of a large collection of melanoma cell lines which have been extensively characterised for HLA genotype and surface expression, oncogene and tumour antigen expression, cytokine secretion, surface molecule expression, adhesion to extracellular matrix components, cytokine gene polymorphisms and other factors of interest to immunologists. This enables investigators to search for cells with particular constellations of HLA alleles, tumour antigens, etc., and then request these from the cell bank. This European Searchable Tumour Cell Line and Data Bank (ESTDAB) was established as a Research Infrastructure of the European Commission. For access to the databank and further details, please go to .     

Cell transformation by viruses

Summary This paper describes some current work pertaining to transformation of cells by oncogenic viruses. Part I includes: (1) the effect of a deoxyribonucleic acid (DNA) tumor virus (SV40) on the antigenic characteristics of transformed cells; (2) in vitro and in vivo methods of detecting virus-specific surface antigens; (3) the role that the host cell may play in the expression of virus-coded antigens; and (4) the presence of virus-induced antigens as a possible mechanism of the apparent nononcogenicity of certain virus variants. Part II discusses (1) the physicochemical properties of the nucleic acid of a ribonucleic acid (RNA) tumor virus-the Moloney sarcoma-leukemia virus (MSV-MLV) complex —(2) a preliminary analysis of viral RNA replication in cells transformed by MSV-MLV, and (3) application to human tumors. Supported in part by Research Grant CA 04600 and by Research Contract PH 43-68-678 within the Special Virus-Cancer Program, National Cancer Institute, National Institutes of Health. Recipient of Research Career Development Award 5-K3-CA 38,614 from the National Cancer Institute, National Institutes of Health     

Monolayer culture of human endometrium: Methods of culture and identification of cell types

Summary Monolayer cultures can be established from human endometrial tissue after enzymatic dispersal into isolated glands or single cells. Three cell types that have distinct morphology by light and electron microscopy are observed in the resulting primary cultures. One cell type, an elongated spindle cell, is similar in appearance to fibroblasts derived from other tissues. A second cell type forms colonies of tightly cohesive cells, ranging in shape from oval to polygonal. These cells have typical organelles and junctional complexes characteristic of epithelial cells from the endometrium. The third cell type assumes a pavement-like appearance composed of polygonal cells when viewed by phase contrast microscopy, but lacks distinctive ultrastructural features of epithelial cells. These cells in culture resemble the endometrial stromal cell, the predominant cell type of the human endometrium in vivo. The epithelial cell does not survive subculturing but the other two cell types can be passaged through several generations and can be stored in liquid nitrogen and subsequently returned to culture. This work was supported by contract N01-CP75956 and grant R01-CA31733 from the National Cancer Institute. V. A. Varma is a recipient of an American Cancer Society fellowship; B. H. Dorman, a predoctoral fellowship from the Chemical Industry Institute of Toxicology; J. M. Siegfried, a training grant (CA09156) from the National Cancer Institute; and D. G. Kaufman, a Research Career Development Award (K04-CA-00431) from the National Cancer Institute.     

Morphological aspects of human lymphoid cells grown in vitro

Summary An established immunoglobulin-producing human cell line exhibits a variety of morphological entities described as reticular, lymphoid, and plasmacytoid cells. Transitional forms are frequent, and time lapse photomicrography experiments demonstrate morphological interconversion. The round forms and their morphological precursors synthesize immunoglubulin and constitute about 44% of the cell population. This cell line displays a variety of morphological and functional properties assigned to in vivo lymphoid cell series. It is proposed as an in vitro model for the study of lymphoid cells. Supported by Grant CA06939-05 and by Special Research Career Development Fellowship Award 1F03 CA42078 from the United States Public Health Service.     

Morphological aspects of human lymphoid cells grown in vitro

Summary An established immunoglobulin-producing human cell line exhibits a variety of morphological entities described as reticular, lymphoid, and plasmacytoid cells. Transitional forms are frequent, and time lapse photomicrography experiments demonstrate morphological interconversion. The round forms and their morphological precursors synthesize immunoglobulin and constitute about 44% of the cell population. This cell line displays a variety of morphological and functional properties assigned to in vivo lymphoid cell series. It is proposed as an in vitro model for the study of lymphoid cells Supported by Grant CA06939-05 and by Special Research Career Development Fellowship Award 1F03 CA42078 from the United States Public Health Service.     

Sensitivity of cultured pancreatic carcinoma cells toAcinetobacter glutaminase-asparaginase

Summary Cultured human pancreatic carcinoma cells (MIA PaCa-2) have been shown previously to be very sensitive toE. coli l-asparaginase (EC II). The present studies have demonstrated that another enzyme,Acinetobacter glutaminase-asparaginase (AGA) is much more effective in inhibiting cell growth. At the concentration of 0.0025 U/ml of AGA activity the enzyme totally inhibited cell growth, whereas the EC II with the same concentration did not show any effect. The inhibition of cell growth correlated well with inhibition of protein and glycoprotein synthesis. The addition ofl-glutamine at the concentration of 1 mM completely reversed the inhibition of protein synthesis. Similarly, the addition ofl-glutamine at the concentration of 3 mM daily on 3 successive days after adding AGA resulted in significant reversal of growth inhibition. The results of this study indicate that the action of AGA on MIA PaCa-2 is, to a great extent, exerted through itsl-glutaminase activity. This work was supported in part by USPHS Grant CA 19182. Dr. Wu is recipient of Research Career Development Award Grant CA00686 and Dr. Yunis is a Howard Hughes Investigator.     

Autologous and homologous immunofluorescent antibody to established breast cancer cell lines

Summary Indirect immunofluorescence tests were performed on 14 established human breast cancer cell lines using sera from a variety of subjects. Autologous reactions were studied on 10 cell lines, with positive reactions demonstrable in 8. Tests using sera from a randomly selected population of breast cancer patients showed reactivity in 40 to 66% depending on the target cell line used. Reactivity to other nonbreast cancer cell lines was rare. Several control populations were tested, including normal blood bank donors, persons with benign breast disease, and persons with other forms of cancer; immunofluorescent antibody was detected much less frequently in sera from these populations than those from the breast cancer group. Positive reactions remained in spite of absorption of serum with heterophile antigens, normal human breast tissue, and AB+ red blood cells. Thus established cell lines of human breast cancer possess antigens commonly recognized by sera from breast cancer patients. This work was supported in part by Grant CA 16789 from the National Cancer Institute, NIH, Department of Health, Education and Welfare.     

Immunomodulatory effects of systemic low-dose recombinant interleukin-2 and lymphokine-activated killer cells in humans

Summary The adoptive immunotherapy of human cancer using lymphokine-activated killer (LAK) cells in combination with high-dose systemic recombinant interleukin-2 (rIL-2) has been associated with global changes in several hematological and immunological parameters while imposing profound toxicity on patients. We have evaluated an alternative LAK cell therapy utilizing low-dose systemic rIL-2 in 27 consecutive patients with metastatic cancer. We report that the administration of systemic low-dose rIL-2 is also characterized by significant changes in immunological and hematological parameters, which are qualitatively similar to those induced by high-dose rIL-2. Low-dose systemic rIL-2, given by i.v. bolus, is cleared to baseline levels within 240 min of administration. The induction of lymphocytosis and eosinophilia, which has characterized other protocols, is also a feature of this protocol. In addition, low-dose systemic rIL-2/LAK cell immunotherapy results in increased peripheral blood mononuclear cell (PBMC) expression of T-cell activation markers such as OKIa, OKT10 and IL-2 receptor. PBMC sampled approximately 100 h after the final infusion of LAK cells demonstrated a statistically significant increase in their ability to kill natural killer (NK)-sensitive and NK-resistent cell lines such as K562 and Daudi compared to baseline values (P <.05). These data suggest that rIL-2-based immunotherapy using low-dose rIL-2 is capable of inducing quantitative hematological and immunological changes while (in combination with LAK cells) retaining the ability to mediate tumor regressionin vivo. Dr. Eberlein was a recipient of an American Cancer Society Career Development Award. This work is supported in part by NIH Grant CA-40555 and the Clinical Research Center Grant 20-9299     

Quantitation of human mammary epithelial antigens in cells cultured from normal and cancerous breast tissues

Summary A sensitive radioimmunoassay technique was developed to quantitatite the level of human breast celltype specific antigens on cells from normal breast and from various established cell lines of breast and nonbreast origins. Polyacrylamide gel electrophoresis revealed four major proteinaceous components (150,000; 75,000; 60,000; and 48,000) in human milk fat globule membranes that were used to immunize rabbits in order to elicit antimammary epithelial cell antibody. Antisera obtained were rendered specific by abosorptions and were able to recognize three specific mammary epithelial components of the breast epithelial cell. Human mammary epithelial (HME) antigen expression was highest (1290 ng/10⁶ cells) in normal breast epithelial cells from primary cultures of normal breasts. Lower levels (range: 955 to 330 ng/10⁶ cells) were found in breast epithelial cells from cell lines established from cancerous breast tissue. Cells of nonbreast origins as well as fibroblasts from breast gave much lower values (less than 30 ng/10⁶ cells). On treatment, with trypsin, of two breast epithelial cell lines (MDA-MB-157 and MCF-7) 80 to 85% of their HME antigen expression was lost, suggesting that a majority of these breast antigens reside on the cell surface. This work was Supported by Grant PTD-99 from the American Cancer Society, Grant CA19455 and CA20286 from the National Cancer Institute, and Biomedical Research Support Grant RR05467 from the National Institutes of Health. Most cells used in the present study were produced with support from National Cancer Institute Contract Y01-CP8-0500, Biological Carcinogenesis Branch, Division of Cancer Cause and Prevention, under the auspices of the Office of Naval Research and the Regents of the University of California.     

Growth of human malignant lymphoid cell lines in serum-free medium

Summary Human T lymphoid cell lines (MOLT-4f, MOLT-3, HSB-2, CEM) and human B lymphoid cell lines (BJAB, RAJI, WIL-2) were grown longterm (up to 8 months) in serum-free medium. This medium consisted of Iscove's modified Dulbecco's medium (IMDM), supplemented with bovine serum albumin (BSA) and transferrin (TF). This serum-free medium containing albumin and transferrin is designated AT-IMDM. Lipids were not essential. Cell viability remained high, greater than 80%, in the serum-free medium and the cells maintained their distinctive characteristics. Interleukin-2 (IL-2) production capacity was maintained by the human T lymphoid cell lines JURKAT-77 and MO in short term culture. This simple medium composed of relatively inexpensive and readily available components should be useful for studies of lymphoid cell growth and differentiation and lymphoid cell products. This research was supported by a grant from the National Institutes of Health, CA 12800, and the Concern Foundation of Los Angeles, and CA 09120 (C. U.)     

Immunohistochemical localization of thyrotropin-releasing hormone (TRH) in skin of Rana pipiens

Summary Using immunofluorescent techniques thyrotropin releasing hormone (TRH) is demonstrated in skin of Rana pipiens and R. catesbeiana. The immunofluorescent-TRH is localized in all cell layers of the epidermis and in the epithelium lining the various cutaneous glands, but not in the dermal layer.We wish to thank Dr. Ronald DeLellis and Ms. Mary Blount for their expert advice and guidance in the immunohistochemical techniques.This investigation was supported by NIH National Research Service Award # 1F32 AMO6018-01 from the NIAMDD to Janice L. Bolaffi and NIH Grant AM 21863 to Ivor M.D. Jackson.     

Adaptation of human long-term B lymphoblastoid cell lines to chemically defined,serum-free media

Summary Attempts were made to adapt human long-term B lymphoblastoid cell lines to prolonged growth in serum-free, chemically defined media. A newly described medium, which is an enriched modification of Dulbecco’s modified Eagle’s medium containing additional amino acids and vitamins, was used. The serum is totally replaced by albumin, transferrin, and soybean lipid. The cell lines were all adaptable from RPMI 1640 over a period of time during which the 10% fetal bovine serum (FBS) concentration was reduced and then eliminated in successive steps. After 3 to 6 wk minor alterations in cell shape and adhesion were noted without significant histological changes. Growth characteristics were comparable in the new medium provided a double initial inoculum was used. A panel of cell surface markers, including surface immunoglobulins, Ia antigens, Fc and complement receptors, and T and B erythrocyte rosettes, all showed no altered expression. Molecular genotyping of Ia antigens was carried out by 2-D gel electrophoresis. The antigens showed their full polymorphism without change and were shed into the new culture medium without alteration. Chromosome analysis was performed on Q-banded karyotypes from one of the lines and showed no alteration resulting from the change to serum-free conditions. Thus long-term B lymphoblastoid cell lines can be adapted to prolonged growth in serum-free medium. This will facilitate the assay and isolation of cell products regulating lymphocyte function and the identification and characterization of cell surface molecules free of interference from undefined serum components. This work is supported by grants from the Medical Research Council of Canada, the National Cancer Institute of Cancer, and the Alberta Heritage Fund.     

Cell Cycle Arrest Drastically Extends the Duration of Gene Silencing After Transient Expression of Short Hairpin RNA

We thank Dr. Didier Trono, Dr. Luk Van Parijs, and Dr. Preet Chaudhary for the gifts of plasmids, Dr. Bey-Dih Chang for cell lines and helpful discussions, and Dr. James Mittler and Dr. Chang Lim for assistance with flow cytometry, and Jennifer Huntington for assistance with some experiments. This work was supported by NIH grant R33 CA95996 (I.B.R.).     

Evidence for two genetic complementation groups in pyruvate carboxylase-deficient human fibroblast cell lines

We have examined genetic complementation in pyruvate carboxylase deficiency by comparing the enzyme activity in polyethylene glycol-induced heterokaryons with that in unfused mixtures of fibroblasts from three affected children. Complementation, manifested as a three- to sevenfold increase in pyruvate carboxylase activity, was observed in fusions between a biotin-responsive multiple carboxylase (pyruvate carboxylase, propionyl CoA carboxylase, and -methylcrotonyl CoA carboxylase) deficient fibroblast line and two other lines deficient only in pyruvate carboxylase activity. Kinetic analysis of complementing pyruvate carboxylase deficient lines, measured by the rate of restoration of enzyme activity as a function of time, revealed that maximum restoration was achieved within 10–24 hr after fusion. This profile is similar to those observed for fusions between the multiple carboxylase deficient line and two lines deficient in propionyl CoA carboxylase activity that are known to represent different gene mutations. Although the patients with pyruvate carboxylase deficiency had similar clinical findings, our studies indicate that pyruvate carboxylase deficiency is genetically heterogeneous, with at least two distinct, probably intergenic, complementation groups.This work was supported by an NIH research grant (AM 25675) and an A. D. Williams research grant (6-48360). B. Wolf is the recipient of an NIH Research Career Development Award (AM 00677) and is aided by a Basil O'Connor Starter Research Grant from The National Foundation-March of Dimes (5-263). G. Feldman is the recipient of an NIH predoctoral training grant (GM 07492). This article is No. 100 from the Department of Human Genetics at the Medical College of Virginia.     

Adenylate kinase deficiency and malignant hyperthermia

Summary In a report of two patients who died of malignant hyperthermia, muscle adenylate kinase deficiency was identified in the father and brother of the deceased. To determine if this enzyme deficiency was a biochemical marker for susceptibility to malignant hyperthermia, we measured adenylate kinase in muscle of three survivors of malignant hyperthermia (MH) and five relatives of survivors of MH attacks with positive caffeine contracture tests. Neither the activity nor the electrophoretic mobility of adenylate kinase differed from four control values. The results show that muscle adenylate kinase deficiency is not a biochemical abnormality shared by all individuals susceptible to malignant hyperthermia.This work has been supported by grants from Muscular Dystrophy Association of America, NIH (NS 11766)Dr. Cerri is recipient of a postdoctoral fellowship from Muscular Dystrophy association and Dr. Willner is recipient of a Teacher Investigator Award from NINCDS     

Effect of room fluorescent light on the deterioration of tissue culture medium

Summary A major cause of tissue culture medium deterioration is exposure to room fluorescent light. Riboflavin and tryptophan present in Dulbecco's modified Eagle's minimum essential medium, when exposed to light, yield toxic photoproducts responsible for loss of the ability of the medium to support clonal growth of human, mouse and Chinese hamster cell lines. Procedures for minimizing medium deterioration are discussed. This work was supported by American Cancer Society Research Grant No. VC-100B and US PHS Research Career Development Award No. 5 K04 GM70537 from the National Institute of General Medical Sciences.